Antiviral composition comprising glycine amide

ABSTRACT

Embodiments relate to the discovery that certain tripeptide amides and glycine amide can be used to inhibit viral infection, including human immunodeficiency virus (HIV) infection. More specifically, medicaments comprising said tripeptide amides and/or glycine amide and methods of using said compounds for the prevention and treatment of viral infection, such as HIV infection, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/235,158, filed Sep. 03, 2002, now abandoned which claims priority toU.S. Provisional Application No. 60/323,650, filed Sep. 19, 2001. Thisapplication claims priority to U.S. application Ser. No. 10/235,158 andU.S. Provisional Application No. 60/323,650, both of which are herebyexpressly incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the discovery that certain tripeptideamides and glycine amide can be used to inhibit viral infection,including human immunodeficiency virus (HIV) infection. Morespecifically, medicaments comprising these tripeptide amides and glycineamide and methods of using said compounds for the prevention andtreatment of viral infection, such as HIV infection, are provided.

BACKGROUND OF THE INVENTION

All viruses are composed of a protein shell surrounding a nucleic acidcontaining core. The protein shell directly surrounding the viralnucleic acid is called a capsid, whereas, the complete protein-nucleicacid complex having both the capsid and the nucleic acid is called anucleocapsid. Arenaviruses, rotaviruses, orbiviruses, retroviruses(including lentiviruses), papillomaviruses, adenoviruses, herpesviruses,paramyxovirus, myxovirus, and hepadnaviruses all exhibit these generalstructural features. (Virology, Fields ed., third edition,Lippencott-Raven publishers, pp 1513, 1645, 1778, 2047, 2113, 2221, and2717 (1996)).

The capsid is composed of many subunits (capsomeres) and capsomeres areformed from several homo- or hetero-polymers of protein. The noncovalentbonds between capsomeres in a viral assembly are of the same sort thatstabilize a folded protein domain. The interface between two subunitscan look very much like a single domain, with amino acid side chainstightly packed against one another. A common feature to most of thevirus structures analyzed is the way in which a polypeptide chain fromone capsomere can extend under or over domains of neighboringcapsomeres. These extended polypeptide arms intertwine with otherpolypeptide arms and help to stabilize the capsid by initiatinghydrophobic interactions, hydrogen bonding, and salt bridges. Contactsbetween individual capsomeres, and for some viruses also contacts withcore proteins, determine the overall capsid structure and if a number ofidentical capsomeres are involved, repeated contacts occur and theresulting structure is symmetrical. (Id. at 62).

Some simple viruses form spontaneously from their dissociated componentswhile others require enzyme-catalyzed modifications of the capsomeres totrigger assembly. Viral self assembly is driven by the stability of theinteractions between protein subunits under conditions that favorassociation. More complex viruses are often constructed fromsubassemblies that have undergone self assembly processes. (Id. at pp62, 70, 1646 and 1888). Although the capsids of many viruses differ inprotein composition, a general viral structural design has evolvedcharacterized by polymerized capsomeres that, in turn, are composed ofseveral homo- or hetero- polymers of protein.

HIV is the name given to a lentivirus that infects humans and thatcauses acquired immuno-deficiency syndrome (AIDS). The lentivirusisolates from humans are grouped into one of two types (HIV-1 and HIV-2)on the basis of serologic properties and sequence analysis ofmolecularly cloned viral genomes. Genetically distinct lentiviruses havebeen obtained from several non-human primate species including Africangreen monkeys, sooty magabeys, mandrills, chimpanzees, and sykes.Collectively, the lentivirus isolates from non-human primates are calledSIV. Sequence analysis reveals that the genomes of some SWV strains andHIV-1 and HIV-2 strains exhibit a high degree of homology. Further,electron microscopy reveals that the ultrastructure of HIV and SIV aresimilar in that both have virions about 110 nm in diameter with acone-shaped nucleocapsid surrounded by a lipid bilayer membrane thatcontains envelope glycoprotein spikes. (Id. at pp.1882–1883).

HIV is a complex retrovirus containing at least seven genes. The viralstructural genes, designated gag, pol, and env, respectively code forthe viral core proteins, reverse transcriptase, and the viralglycoproteins of the viral envelope. The remaining HIV genes areaccessory genes involved in viral replication. The gag and env genesencode polyproteins, i.e., the proteins synthesized from each of thesegenes are post-translationally cleaved into several smaller proteins.

Although the overall shape of HIV and SIV virions is spherical, thenucleocapsid is asymmetrical having a long dimension of about 100 nm, awide free end about 40–60 nm, and a narrow end about 20 nm in width. Thenucleocapsid within each mature virion is composed of two molecules ofthe viral single-stranded RNA genome encapsulated by proteinsproteolytically processed from the Gag precursor polypeptide. Cleavageof the gag gene polyprotein Pr55^(gag) by a viral coded protease (PR)produces mature capsid proteins. These gag gene products are the matrixprotein (p17), that is thought to be located between the nucleocapsidand the virion envelope; the major capsid protein (p24), that forms thecapsid shell; and the nucleocapsid protein (p9), that binds to the viralRNA genome. This proteolytic processing in infected cells is linked tovirion morphogenesis. (Id. at pp 1886–1887).

The major capsid protein p24 (also called CA) contains about 240 aminoacids and exhibits a molecular weight of 24–27 kD. The protein p24self-associates to form dimers and oligomeric complexes as large asdodecamers. Genetic studies with mutations in the HIV-1 gag polyproteinhave identified several functional domains in the p24 protein includingthe C terminal half of the molecule and a major homology region (MHR)spanning 20 amino acids that is conserved in the p24 proteins of diverseretroviruses. These mutations appear to affect precursor nucleocapsidassembly. (Id. at pp 1888–1889).

Since the discovery of HIV-1 as the etiologic agent of AIDS, significantprogress has been made in understanding the mechanisms by which thevirus causes disease. While many diagnostic tests have been developed,progress in HIV vaccine therapy has been slow largely due to theheterogeneous nature of the virus and the lack of suitable animalmodels. (See, e.g., Martin, Nature, 345:572–573 (1990)).

A variety of pharmaceutical agents have been used in attempts to treatAIDS. Many, if not all, of these drugs, however, create serious sideeffects that greatly limit their usefulness as therapeutic agents. HIVreverse transcriptase is one drug target because of its crucial role inviral replication. Several nucleoside derivatives have been found toinhibit HIV reverse transcriptase including azidothymidine (AZT,zidovudine®). AZT causes serious side effects such that many patientscannot tolerate its administration. Other nucleoside analogs thatinhibit HIV reverse transcriptase have been found to cause greater sideeffects than AZT. Another drug target is the HIV protease (PR) crucialto virus development. PR is an aspartic protease and can be inhibited bysynthetic compounds. (Richards, FEBS Lett., 253:214–216 (1989)).Protease inhibitors inhibit the growth of HIV more effectively thanreverse transcriptase inhibitors but prolonged therapy has beenassociated with metabolic diseases such as lipodystrophy,hyperlipidemia, and insulin resistance.

Additionally, HIV quickly develops resistance to nucleoside/nucleotideanalogue reverse transcriptase inhibitors and protease inhibitors. Thisresistance can also spread between patients. Studies have shown, forexample, that one tenth of the individuals recently infected by HIValready have developed resistance to AZT, probably because they wereinfected by a person that at the time of transmission carried a virusthat was resistant to AZT.

It would be useful in the treatment and prevention of viral infections,including HIV and SIV, to have specific and selective therapeutic agentsthat cause few, if any, side effects.

SUMMARY OF THE INVENTION

The present invention is related to molecules that inhibit viralinfectivity, specifically replication of Human Imnmunodeficiency Virus(HIV). It was discovered that certain tripeptides and the amino acidglycine, with their carboxyl terminus hydroxyl group replaced with anamide group, have an inhibiting effect on the replication of viruses,such as HIV. It is contemplated that these molecules inhibit viralreplication by affecting protein-protein interactions during capsidassembly and/or by interfering with virus budding.

In addition to glycine amide (G-NH₂), the tripeptide amides AIG-NH₂,GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂ are the preferred species. These molecules andpeptidomimetics resembling their structure (collectively referred to as“peptide agents”) are used in a monomeric or multimeric form. Glycineamide and the tripeptide amides (i.e., peptide agents) are suitable fortherapeutic and prophylactic application in mammals, including man,suffering from viral infection. Glycine amide or any one of AIG-NH₂,GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂ can be administered individually or themolecules can be provided in any combination (e.g., glycine amide can beprovide with GLG-NH₂ or APG-NH₂ can be provided with GFG-NH₂ etc.)

In one embodiment, a composition for inhibiting viral replication inhost cells infected with a virus has an effective amount of glycineamide and/or a peptide in amide form selected from the group of AIG-NH₂,GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂. In some embodiments, the compositions describedabove are joined to a support and in other embodiments, the compositionsdescribed above are incorporated into a pharmaceutical having apharmaceutically acceptable carrier.

Methods of inhibiting viral replication in a host cell are alsoembodiments of the present invention. One approach, for example,involves administering to a cell an effective amount of glycine amideand/or a peptide in amide form selected from the group consisting ofAIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂. The method described above can be supplementedwith an antiviral treatment selected from the group consisting ofnucleoside analogue reverse transcriptase inhibitors, nucleotideanalogue reverse transcriptase inhibitors, non-nucleoside reversetranscriptase inhibitors, and protease inhibitors. The glycine amideand/or the tripeptide amide used in the method above can be joined to asupport or can be administered in a pharmaceutical comprising apharmaceutically acceptable carrier.

In another embodiment, a composition for inhibiting HIV replication inhost cells includes an effective amount of glycine amide and/or apeptide in amide form selected from the group consisting of AIG-NH₂,GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂. In some embodiments, the glycine amide or thetripeptide amides are joined to a support and in other embodiments,these molecules are incorporated into a pharmaceutical comprising apharmaceutically acceptable carrier.

In another method, an approach to inhibit HIV replication in host cellsis provided, which involves administering to said cells an effectiveamount of glycine amide and/or a peptide in amide form selected from thegroup consisting of peptides of the formula AIG-NH₂, GFG-NH₂, GWG-NH₂,FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. Thismethod can also be supplemented by an antiviral treatment selected fromthe group consisting of nucleoside analogue reverse transcriptaseinhibitors, nucleotide analogue reverse transcriptase inhibitors,non-nucleoside reverse transcriptase inhibitors, and proteaseinhibitors. Further, the glycine amide and/or tripeptide amide used inthis method can be joined to a support or can be administered in apharmaceutical comprising a pharmaceutically acceptable carrier.

In another method, an approach for interrupting viral capsid assembly isprovided. This approach involves contacting a cell with an effectiveamount of glycine amide and/or a peptide in amide form selected from thegroup consisting of peptides of the formula AIG-NH₂, GFG-NH₂, GWG-NH₂,FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. Theglycine amide and/or the tripeptide amide can be joined to a support orincorporated in a pharmaceutical.

In another method, an approach for inhibiting proper viral budding isprovided. This approach involves contacting a cell with an effectiveamount of glycine amide and/or a peptide in amide form selected from thegroup consisting of peptides of the formula AIG-NH₂, GFG-NH₂, GWG-NH₂,FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. Theglycine amide and/or the tripeptide amide can be joined to a support orincorporated in a pharmaceutical.

In still another method, an approach for interrupting HIV capsidassembly is provided. This approach also involves contacting a cell withan effective amount of glycine amide and/or a peptide in amide formselected from the group consisting of peptides of the formula AIG-NH₂,GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂. The glycine amide and/or the tripeptide amideof this method can be joined to a support or incorporated in apharmaceutical.

In still another method, an approach for inhibiting proper HIV buddingis provided. This approach also involves contacting a cell with aneffective amount of glycine amide and/or a peptide in amide formselected from the group consisting of peptides of the formula AIG-NH₂,GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂. The glycine amide and/or the tripeptide amideof this method can be joined to a support or incorporated in apharmaceutical.

Methods of identification of peptide agents that inhibit viralreplication, specifically HIV replication are also provided. By onemethod, for example, a peptide agent for incorporation into ananti-viral pharmaceutical is identified by contacting a plurality ofcells infected with a virus with an effective amount of a peptide agent,analyzing the virus for incomplete capsid formation or impaired viralbudding, and selecting the peptide agent that induces incomplete capsidformation or induces impaired viral budding. This method can involve ananalysis of capsid formation or viral budding that employs microscopy(e.g., electron microscopy) and the virus can be selected from the groupconsisting of HIV-1, HIV-2, and SWV. Further, the peptide agentidentified can be selected from the group consisting of glycine amide, atripeptide amide, and a peptidomimetic resembling glycine amide or atripeptide amide. For example, the peptide agent above can be selectedfrom the group consisting of glycine amide, AIG-NH₂, GFG-NH₂, GWG-NH₂,FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂.

In another embodiment, a method of identifying a peptide agent thatbinds to a viral protein is provided. Some aspects of this methodinvolve providing a viral protein, contacting the viral protein with aneffective amount of a peptide agent, and detecting the formation of acomplex comprising the viral protein and the peptide agent. Some methodsuse a viral protein that is from a virus selected from the groupconsisting of HIV-1, HIV-2, and SIV. Further, in some embodiments, thepeptide agent is selected from the group consisting of glycine amide, atripeptide amide and a peptidomimetic resembling glycine amide or atripeptide amide. Desirably, the method above employs glycine amideand/or a peptide agent selected from the group consisting of AIG-NH₂,GFG-NF₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂. Additionally, a method of making apharmaceutical is provided in which the peptide agent identified by themethods above are incorporated in a pharmaceutical.

Another approach to making a pharmaceutical involves administering to acell, especially a cell present in an animal such as a human, aneffective amount of glycine amide or a peptide in amide form, describedabove, detecting an inhibition of viral replication in the cell, andincorporating the molecule that causes inhibition of viral replicationinto the pharmaceutical. This method can involve the use of glycineamide and/or a tripeptide amide selected from the group consisting ofAIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂. Further, this method can be supplemented withadministration of an antiviral compound selected from the groupconsisting of nucleoside analogue reverse transcriptase inhibitors,nucleotide analogue reverse transcriptase inhibitors, non-nucleosidereverse transcriptase inhibitors, and protease inhibitors into thepharmaceutical. Additionally, the method above can be supplemented byincorporating a carrier into the pharmaceutical.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of GPG-NH₂ and ALG-NH₂ analogues on HIV-1replication in H9 cells. The p24 levels in cell culture supernatants ofHIV-1 infected cells, cultured in the presence or absence oftripeptide-amides, were measured at day 7 after infection by antigencapture-enzyme-linked immunosorbent assay (ELISA).

FIG. 2 shows the effect of glycine-amide and GPG-NH₂ and ALG-NH₂analogues on HIV-1 replication in CEM cells. The p24 levels in cellculture supernatants of HIV-1 infected cells, cultured in the presenceor absence of glycine amide or tripeptide-amides, were measured at day11 after infection by antigen capture enzyme-linked immunosorbent assay(ELISA).

FIG. 3 shows H9 cells that were infected with 100 TCID₅₀ of HIV-1 in thepresence or absence of 100 μM of GPG-NH₂ or one of its metabolites. Cellsupernatants were harvested at day 11 post infection and the levels ofp24 were measured by p24 antigen capture enzyme-linked immunosorbentassay (ELISA). 1=Infected control; 2=GPG-OH (negative control);3=non-infected control; 4=GPG-NH₂; 5=G-NH₂; 6=GP-OH; 7=G-OH; 8=PG-NH₂.

FIG. 4 shows the dose dependent inhibition of HIV-1 replication in H9cells in the presence of GPG-NH₂ (white triangles) or G-NH₂ (blacksquares), as measured by p24 levels in cell culture supernatant at day11 after infection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

It has been discovered that glycine amide and certain tripeptide amidesprevent and/or inhibit viral infection. Such amino acid or peptides areuseful in the treatment of viral disease, particularly in HIV/AIDSafflicted subjects, and as preventive agents for patients at-risk ofviral infection, particularly HIV infection, and for use with medicaldevices where the risk of exposure to virus is significant.

The disclosure below demonstrates that glycine amide and certaintripeptides in amide form, such as AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂,GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂ inhibit thereplication of viruses, for example HIV-1. Evidence of the inhibition ofviral replication was found in viral infectivity assays that monitor theamount of capsid protein present in culture supernatant.

Several approaches to making biotechnological tools and pharmaceuticalcompositions comprising glycine amide and/or tripeptide amides andpeptidomimetics that resemble these molecules (collectively referred toas “peptide agents”) are given below. Preferred peptide agents areglycine and tripeptides with an amide group at their carboxy termini,and include the following: G-NH₂, AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂,GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. In someembodiments, the peptide agents are provided in monomeric form; inothers, the peptide agents are provided in multimeric form or inmultimerized form. Support-bound peptide agents are also used in severalembodiments.

Pharmaceutical compositions comprising peptide agents are administeredas therapeutics or prophylactics or both for the treatment and/orprevention of viral disease, particularly, HIV infection. In someembodiments, the pharmaceutical compositions comprising peptide agentsare administered in combination with other antiviral treatmentsincluding nucleoside analogue reverse transcriptase inhibitors,nucleotide analogue reverse transcriptase inhibitors, non-nucleosidereverse transcriptase inhibitors, and protease inhibitors. These smallmolecules are resistant to acid hydrolysis. A significant amount oftripeptide amides, for example, is effectively delivered to blood,plasma, and organ tissue when administered to test subjects. Theadministration of large doses of small peptides to test subjects isrelatively nontoxic. (See U.S. Pat. No. 6,258,932, which is hereinexpressly incorporated by reference in its entirety).

Additionally, several methods of identifying a peptide agent thatinhibits or prevents viral replication or interrupts viral capsidassembly or both are provided. By one approach, an effective amount of apeptide agent is contacted with cells infected with a virus and thecells are analyzed for viral replication or the presence of viralproducts. Accordingly, a capsid protein (e.g., p24) is contacted with apeptide agent, for example a peptide in amide form, as described above,and a complex comprising the capsid protein (e.g., p24) bound with thepeptide agent is identified.

The amide form of the molecules listed in TABLE 1 were tested. Many ofthese molecules were selected and synthesized because they aremodifications of sequences that correspond to HIV and/or SIV viralproteins. The tripeptide amides of TABLE 1 were synthesized according tothe method disclosed in EXAMPLE 1 below, but could of course besynthesized by any method known in the art. Glycine amide was purchasedfrom Bachem, Switzerland (product No. 4025766), whereas Glycine-OH waspurchased from Merck, Germany (product No. 14201-250). GPG-NH₂ was alsopurchased from Isochem, France.

TABLE 1 GPG-NH₂: glycyl-prolyl-glycine-amide ALG-NH₂:alanyl-leucyl-glycine-amide GFG-NH₂: glycyl-phenylalanyl-glycine-amideGWG-NH₂: glycyl-tryptophanyl-glycine-amide FLG-NH₂:fenylalanyl-leucyl-glycine-amide GYG-NH₂: glycyl-tyrosyl-glycine-amideAPG-NH₂: alanyl-prolyl-glycine-amide GLG-NH₂:glycyl-leucyl-glycine-amide α-t-butylglycine-PG-NH₂:α-tertiary-butylglycine-prolyl-glycine-amide LNF-NH₂:leucyl-asparagyl-phenylalanine-amide AIG-NH₂:alanyl-isoleucyl-glycine-amide GGG-NH₂: glycyl-glycyl-glycine-amidePGR-NH₂: prolyl-glycine-arginine-amide G-NH₂: glycine amide

EXAMPLE 1

In this example, the approaches used to obtain the tripeptide amideslisted above are disclosed. The tripeptide amides were chemicallysynthesized with an automated peptide synthesizer (Syro, Multisyntech,Witten, Germany) largely according to the manufacturer's instructions.The synthesis was run using 9-fluorenylmethoxycarbonyl (Fmoc) protectedamino acids (Milligen, Bedford, Mass.) according to standard protocols.The modified peptides were created by substituting an amino group forthe hydroxyl residue normally present at the terminal carboxyl group ofa peptide. That is, instead of a terminal COOH, the peptides weresynthesized to have CO—NH₂. For example, in addition to glycine amide,the preferred tripeptide amides include AIG-NH₂, GFG-NH₂, GWG-NH₂,FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. TABLE 2lists the Fmoc amino acids used.

TABLE 2 Fmoc-Ala-OH Fmoc-Arg(Pbf)-OH Fmoc-Asn(Trt)-OH Fmoc-Asp(OtBu)-OHFmoc-Cys(Trt)-OH Fmoc-Glu(OtBu)-OH Fmoc-Gln(Trt)-OH Fmoc-Gly-OHFmoc-His(Trt)-OH Fmoc-Ile-OH Fmoc-Leu-OH Fmoc-Lys(Boc)-OH Fmoc-Met-OHFmoc-Phe-OH Fmoc-Pro-OH Fmoc-Ser(tBu)-OH Fmoc-Thr(tBu)-OHFmoc-Trp(Boc)-OH Fmoc-Tyr(tBu)-OH Fmoc-Val-OH

Rink amide MBHA resin (MultiSynTech, Witten, Germany) was used. Otherreagents used to prepare the tripeptide amides included Acetic acid,Acetic anhydride, 2-(1H-Benzotriazole-1-yl)-1,1,3,3, tetramethyluroniumtetrafluoroborate (TBTU), Diisopropylcarbodiimide (DIC),Dimethylformamide (DMF), Ethanedithiol (EDT), Ether,Ethyldiisopropylamine (DIPEA), Hydroxybenzotriazole (HOBt), Isopropanol,Lithium chloride, Methanol, Methylphenylsulfide, 1-Methyl-2-pyrrolidone(NMP), Piperidine, Pyridine, and Trifluoroacetic acid (TFA), all ofwhich can be obtained from a variety of commercial suppliers.

The peptide synthesis was conducted as follows. The peptide sequenceswere entered into the synthesizer computer using the amino acid oneletter codes and the correct sequences were verified by printing theentry. Next, the “cycle/chemfile relation”, which specifies a chemfilefor each coupling cycle, was initiated. Then the “chemfile editor” wasinitiated to study or modify the chemfiles, if necessary. A separatechemfile, which begins and ends with an Fmoc-deprotection step, was usedfor the first coupling cycle. The chemfiles used for the other couplingcycles only end with an Fmoc-deprotection. The chemfiles “swell singlecoupling DIC” (cycle 1) and “single coupling DIC” (cycle 2->) were usedfor standard syntheses.

Once the chemfiles and sequences had been entered, the “calculation”phase was begun. The resin loading equivalents (excess of amino acids)and resin amount weighed in each reactor was entered. A calculationreport was printed. The resin in the plastic reactors was weighed andplaced in the reaction block. A stock solution of HOBt in fresh DMFcontaining molecular sieve was prepared. The amino acids were weighed in50 ml tubes and the HOBt solution above was used to dissolve the aminoacids according to the calculation report. The molar relation betweenamino acid and HOBt was 1:1. The addition of HOBt prevented amino acidracemization. The tubes containing the amino acid solutions were thentransferred to the building block box. The amino acid tubes were placedin alphabetical order from left to right according to the amino acidone-letter codes. The amino acid positions were also specified on thelid of the building block box and on the computer window of the SYRO IIprogram.

Bottle 3 from the robot table was removed and washed with fresh DMF.Bottle 3 was used to dissolve DIC in DMF according to the calculationreport then it was placed back into position on the robot table. DICactivates the carboxy group of the amino acids. Bottles 1 and/or 2 wasused to prepare a 40% solution of piperidine in DMF according to thecalculation report. It was not necessary to use fresh DMF for thepiperidine solutions. The bottles (1 and 2) used for 40% piperidine wereidentified as such in the chemfiles. Piperidine cleaves theamino-protecting Fmoc group before each coupling. The chemfile used forcoupling cycle 1 contained two piperidine cycles since an additionalFmoc group had to be removed from the resin before coupling of the firstamino acid. The piperidine bottles were placed back into position on therobot table.

Under some circumstances, the double coupling of amino acids may bedesired. Double coupling may result in more efficient synthesis ofdifficult sequences. The peptide quality may also be improved byincreasing the coupling time and temperature or by increasing the Fmocdeprotection time. However, longer coupling times at higher temperaturesmay lead to unwanted side reactions and peptide degradation. Normally,the amino acids are coupled 40–60 minutes at 30° C. DIC is used as anactivator in standard synthesis. During double coupling, DIC may be usedas the only activator, or it may be used only in the first coupling inconjunction with a second activator system used in the second coupling.The use of different modes of activation may further increase thecoupling efficacy. A second activator is TBTU together with DIPEA.However, this activator system has limited solubility. The use of doublecoupling and different activators is specified in the chemfiles.Solutions of DIPEA and TBTU in NMP are prepared in bottles 4 and 5according to the calculation report. Before preparing the solutions, thebottles were washed with fresh NMP.

After each double coupling the unreacted free amino groups can beblocked by acetylation (capping). Acetylation prevents elongation ofdeletion peptides missing one or several amino acids. Also, theacetylated peptides are usually easily separated from the correctsequence since they appear late in reverse phase HPLC chromatogram dueto their hydrophobicity. The acetylation solution 10% aceticanhydride/5% pyridine in DMF was prepared in bottle 7. Before preparingthe acetylation solution, the bottle was washed with fresh DMF. Somecrystals of lithium chloride were also added to the acetylationsolution.

Next, the robotic arms and the brass rods were cleaned with a cloth thatwas wetted with isopropanol once the synthesizer had been turned off.Then the synthesizer was turned back on and the reagent bottles andbuilding block box were placed in their fixed positions on the robottable. A 10 L brown glass bottle was then filled with DMF that was notolder than two weeks. The gas tube (argon or nitrogen gas) was openedand the gas pressure was regulated with the pressure membrane regulatoron the robot. The pressure was maintained at approximately 1 bar. Thesynthesis was started by clicking on “start synthesis” in the robot menuand the start and end positions were selected. During a large synthesisit may be necessary to fill the 10 L bottle with more DMF. The amount ofDMF was checked regularly during the synthesis.

Once the synthesis had finished, a synthesis report was printed andanalyzed to determine if all the couplings had completed. Next, asuitable cleavage chemfile was selected by clicking on “chemfile editor”in the tools menu. Then the “cycle/chemfile relation” was selected tospecify the cleavage chemfile for coupling cycle 1. New glass tubes wereplaced in the cleavage box rack in the fume hood to the right of thesynthesizer. The lid was placed on the cleavage box and the cleavage wasinitiated by clicking on “start synthesis” in the robot menu. Cycle 1was selected for both start and end positions.

The cleavage solution was then prepared during the washing and transfersteps at the start of the cleavage chemfile. The cleavage mixture 2%water/2% EDTA/2% methylphenylsulfide/94% TFA was transferred to bottle 6and placed in its fixed position on the robot table. All other bottlesand the building block box were removed from the robot table. Thestandard “cleavage aut” chemfile contains several programmed stops toallow manual check of the cleavage line washings and also to allowchange of glass tubes in the cleavage box before transfer of thecleavage mixture from the reactor block. The automatic dispensing ofcleavage mixture was always monitored carefully. In the “cleavage aut”chemfile, cleavage mixture was added twice to reactors and transferredto the glass tubes. The total cleavage time was approximately 3 hoursfrom addition of the first portion of cleavage mixture to the peptidereactors.

The tubes were stirred a few times after the cleavage mixture containingthe peptide had been transferred to the glass tubes. After 3 hours, thecleavage mixtures were transferred from the glass tubes to 15 mlpolypropylene centrifuge tubes with screw caps and labeled with peptidenumbers. Approximately 6 ml of ether was dispensed with the automaticdispenser to the 15 ml tubes. The tubes were capped and gently mixed byhand.

The peptides were precipitated in the ether while the cleavage chemicalsremained soluble. If a peptide did not precipitate immediately, it waskept in the fume hood for 1–3 days and the precipitate slowly developed.The peptides were then centrifuged for 5 min at 4000 rpm at 0° C. Theether was removed, fresh ether was added to the tubes and thepeptide/ether solution was mixed gently again. A pasteur pipette wasused when the peptide adhered to the bottom of the tube. After four suchether washings, the peptides were left to dry in the fume hoodovernight.

After drying overnight, the dried peptides were resuspended inapproximately 3–10 ml milli-Q water. A few drops of concentrated aceticacid was added to neutral and basic peptides that did not readilydissolve in pure water. The dissolved peptides were then transferred to4 ml, 10 ml or 30 ml glass vials, the vials were covered with papercloths held by rubber bands, and the vials were stored at −80° C. for atleast 2 hours before lyophilization. All peptides were lyophilized andthen dissolved at the appropriate concentration in Milli-Q water orphosphate-buffered saline (PBS). The peptides were next analyzed byreverse phase high performance liquid chromatography (RP-HPLC) usingeither a Chromolith Performance RP-18e 100-4.6 column (for analyticalRP-HPLC) or a LiChrospher 100 RP-18e (10 μm) 250-10 (for preparativeRP-HPLC).

RP-HPLC was performed as follows. The D-7000 HPLC system manager (HSM)was initiated, the purge valves of both pumps was opened and the pumpswere purged. The purge flow was run for approximately one minute toflush the tubings. The purge flow was then stopped and the purge valvesof the pumps were closed. In this system, pump A pumped water and pump Bpumped the second solvent (usually methanol). Approximately, 0.25%trifluoroacetic acid was added to all solvents. Initially, a flow of100% water was run through the column.

Next, a suitable method file and sample table was selected. The columnswere equilibrated in water for at least 20 min or until the flow linewas stable. For the analytical runs the following gradient was used: 0min—100% water/0% methanol; 1.3 min—100% water/0% methanol; 6.3 min—0%water/100% methanol; 7.5 min—0% water/100% methanol; 8.8 min—100%water/0% methanol; and 10.0 min—100% water/0% methanol. Flow rate on theanalytical column was 2 ml/min. and approximately 100 μl of sample wasinjected onto the column. Small columns were used for analytical HPLCand fractions were not collected.

For the preparative runs the following gradient was used: 0 min—100%water/0% methanol; 5.0 min—100% water/0% methanol; 25.0 min—0%water/100% methanol; 30.0 min—0% water/100% methanol; 35.0 min—100%water/0% methanol; and 40.0 min—100% water/0% methanol. Flow rate forthe preparative column was 6 ml/min. and approximately 1 ml of samplewas injected onto the column. During preparative HPLC, the fractioncollector was setup to collect one or several sample fractions. The rackparameters were carefully monitored to insure that the rack wascompatible with the auto sampler. Once the HPLC runs were completed,that is, the peptide peak was identified and/or collected, 50% B(methanol) was run through the column (at least five column volumes) tostrip the column. In the disclosure below, several assays that were usedto identify the molecules that inhibit HIV-1 infection are described.

Small Molecules That Inhibit and/or Prevent HIV Replication andInfection

The tripeptide amides made according to EXAMPLE 1 were used in severalHIV-1 infectivity assays to determine the ability of said tripeptideamides to inhibit HIV replication and/or infection. The efficiency ofHIV-1 replication and status of HIV-1 infection was monitored by theconcentration of p24 protein in the cell supernatant. (See e.g., U.S.Pat. Nos. 5,627,035 and 6,258,932, herein expressly incorporated byreference in their entireties, which describe similar HIV infectivityassays and others that can be used to analyze the tripeptide amidesdescribed herein). EXAMPLE 2 describes an approach that was used toscreen several tripeptide amides and glycine amide for the ability toinhibit HIV-1 infection.

EXAMPLE 2

In this example, the methods that were used to analyze the ability ofvarious tripeptide amides and glycine amide to inhibit HIV-1 replicationare disclosed. In a first set of experiments, approximately 3×10⁵ H9cells were infected with HIV-1 (e.g., 50–100 TCID₅₀ per 300,000 H9cells) to test the inhibitory effect of various tripeptide amidesprovided at 100 μM concentration. (See TABLE 3).

By one approach, virus was added at 50–100 TCID₅₀ to 3×10⁵ H9 cells in atotal volume of 500 μl containing RPMI 1640 medium supplemented with 10%(v/v) heat-inactivated fetal bovine serum (FBS), L-Glutamine, andBensylpenicillin and Streptomycin (PS) (approximately 0.5 ml added to500 ml RPMI medium), all available through LifeTechnology/GIBCOlaboratories. This media is referred to as “RPMI++ media.” Cell countingwas accomplished using 0.2% tryphanblue dissolved in PBS and a Bürkercell counter chamber. The virus and cells were then mixed gently on avortex and were incubated at 37° C. for one hour and thirty minutes.Next, the cells were pelleted at 1200 rpm for 7 minutes and thesupernatant was discarded.

The cells were then resuspended in RPMI++ at a concentration of 3×10⁵cells per ml. One ml of cell suspetion was then added to each well in a24-well plate containing the different tripeptide amides or glycineamide in 0.6 ml RPMI++. The final concentration of tripeptide amides orglycine amide was approximately 100 μM. Cells were then incubated at 37°C. in a 5% CO₂ enriched incubator. The medium was changed on day 4, 7,and finally day 11. The infection was stopped on day 11 or 14. Duringeach media change, approximately 0.8 ml was replaced and 0.8 ml of thesupernatant was transferred to a sterile 96 well plate and stored at−80° C. for p24 analysis.

The presence of p24 in the supernatants was determined using a p24antigen detection method. Suitable p24 detection kits are commerciallyavailable (e.g., Abbott Laboratories, North Chicago, U.S.A.). By oneapproach, a capture-assay is employed, wherein the viral antigen iscaptured on a 96-well plate coated with a polyclonal anti-p24 rabbitserum (Swedish Institute for infectious disease control). The capturedantigen is then detected with peroxidase conjugated anti-p24 mousemonoclonal antibodies. The conjugate is a pool of three differentmonoclonal antibodies. The analysis was performed with cell freesupernatant directly from the cell culture or with supernatant that hadbeen stored at −20° C. to −80° C.

Accordingly, a p24 standard was diluted (e.g., 4, 2, 1, 0.5, 0.25,0.125, 0.0625 ng/ml) in RPMI++ media. The standard, recombinant HIV-1LAI gag p24, was purchased from NIBSC, Centralized Facility for AIDSReagent, MRC. (order no EVA620). In some cases, serial dilutions of thesupernatants were made so as to more accurately detect p24concentration. Coated plates (e.g., plates coated with a polyclonalanti-p24 rabbit serum) were washed 4 times with approximately 300–350μl/well washing buffer (PBS with 0.05% Tween-20). The plates wereinverted and tapped against absorbant paper after each wash to discardthe superfluous washing buffer. Approximately, 100 μl of each sample andstandard was added to individual wells on the plate. The plates werecovered with tape and incubated at 37° C. for 2 hours or in the dark atroom temperature over night.

Next, the plates were washed again as above. Approximately 100 μl/wellof conjugate diluted in conjugate buffer (PBS with 0.5% Triton X-100,0.5% Bovine Serum Albumin, 0.05% Tween-20, and 10% Fetal Bovine Serum).The plates were then covered with tape and incubated at 37° C. for 2 to4 hours. The OPD substrate (ABBOTT) was then prepared by adding 1 tabletof OPD per 5 ml OPD substrate solution (12.8 mg OPD (o-phenylenediamine.2 HCl) per tablet and 1 tablet was dissolved in 5 ml citrate-phosphatebuffer containing 0.02% hydrogen peroxidase). The solution was kept inthe dark until it was used. The conjugate bound plates were then washedas above and, after the final wash, 100 μof OPD substrate solution/wellwas added and the plates were incubated at room temperature for 30minutes. The plates were protected from light during this period. Thereaction was stopped with 100 μl 2.5M H₂SO₄/well and the absorbance wasread at 490 nm and 650 nm As discussed in greater detail below, it wasdiscovered that glycine amide and the tripeptide amides AIG-NH₂,GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂ inhibit HIV-1 infection.

The results of the experiments described in EXAMPLE 2 are shown in FIG.1 and TABLE 3. Accordingly, several tripeptide amides were found toinhibit HIV-1 replication in H9 cells at a 100 μM concentration.Although some tripeptide amides had little affect on HIV infectivity(e.g., PGR-NH₂, OPA-NH₂, GhydPG-NH₂, GGG-NH₂, tbutGLG-NH₂, andmetALG-NH₂) many tripeptide amides almost completely inhibited HIVreplication (e.g., AIG-NH₂, FLG-NH₂, GLG-NH₂, GPG-NH₂, α-tbutGPG-NH₂,APG-NH₂, GFG-NH₂, and GWG-NH₂). In this experiment GPG-NH₂ was used as apositive control whereas GGG-NH₂ and PGR-NH₂, which were known to notinhibit HIV replication, were used as negative controls.

TABLE 3 p24 ng/ml Peptides Mean Std. Dev. AIG-NH2 10.69 2.55 metALG-NH249.80 17.88 FLG-NH2 0.25 0.06 GLG-NH2 2.71 0.75 tbutGLG-NH2 48.26 6.51GPG-NH2 0.42 0.08 tbutGPG-NH2 0.44 0.15 APG-NH2 1.13 0.40 GGG-NH2 61.829.95 GFG-NH2 0.45 0.17 GWG-NH2 0.61 0.29 GHydPG-NH2 78.43 18.68 GPA-NH278.88 10.17 PGR-NH2 55.93 7.48 infected control 53.31 7.41

Several tripeptide amides and glycine amide were also evaluated for theability to inhibit HIV replication in infected CEM cells, another T-cellline. The methods described in EXAMPLE 2 were used to conduct theseexperiments and FIG. 2 and TABLE 4 show the results. Again, GFG-NH₂,GWG-NH₂, FLG-NH₂, APG-NH₂, and tbutGPG-NH₂ were found to inhibit HIV-1replication in infected cells at 100 μM concentration. Additionally, itwas discovered that GYG-NH₂, and glycine amide inhibited HIV-1replication in CEM cells. In contrast, an inhibition of HIV-1replication was not observed for the natural amino acid glycine (G-OH).In this experiment LNF-NH₂ which was known to not inhibit HIVreplication was used as negative control.

TABLE 4 Mean p24 Peptides (ng/ml) Std. Dev. GFG-NH2 0.08 0.13 GWG-NH20.15 0.04 FLG-NH2 0.00 0.06 GYG-NH2 0.02 0.02 APG-NH2 0.01 0.05tbut-GPG-NH2 −0.01 0.06 G-NH2 0.04 0.09 G-OH 1.73 0.41 GPG-NH2 0.07 0.09LNF-NH2 1.45 0.79 Inf. control 1.62 0.75 Non-inf. control 0.01 0.03

In another set of experiments the ability of glycine amide to inhibitHIV replication was more closely analyzed. In these studies, HIVinfected H9 cells were cultured in the presence 100 μM GPG-OH, GPG-NH₂,G-NH₂, GP-OH, G-OH, or PG-NH₂, as described previously (see EXAMPLE 2).As shown in FIG. 3, the amount of p24 detected in the culturesupernatant of G-NH₂ treated cells (#5) at day 11 was almost identicalto that found in the non-infected control (#3) and cells treated withGPG-NH₂ (#4). In contrast, HIV infected H9 cells treated with G-OH (#7)had considerable p24 present in the culture supernatant (approximately65 ng/ml). These results provide additional evidence that glycine amidecan be used to inhibit HIV infection or replication.

In the next series of experiments, it was discovered that glycine amideinhibits HIV replication in a dose dependent manner. HIV infected H9cells were cultured in the presence of varying concentrations 0.5–20 μM)of glycine amide or GPG-NH₂ (positive control) and the amount of p24present in the culture supernatant was determined at day 11 afterinfection. The methodology employed was that described in EXAMPLE 2. Asshown in FIG. 4, significant inhibition of HIV replication was achievedat concentrations of glycine amide less than 5 μM. The data also showthat 20 μM glycine amide almost completely inhibited HIV replication.These results clearly indicate that as the concentration of glycineamide was increased the amount of p24 in the supernatant, whichindicates the amount of HIV infection or HIV replication, decreased.

In another series of experiments, it was determined that the tripeptideamides GPG-NH₂ and ALG-NH₂ interfere or inhibit proper budding of HIV.HIV infected cells cultured with GPG-NH₂ or ALG-NH₂, when viewed byelectron microscopy, displayed a unique nodular structure associatedwith the outer membrane of virus producing cells. These structures wereonly found in cells treated with GPG-NH₂ or ALG-NH₂. Most of the treatedcells that carried virus particles had these nodular structures (74%with GPG-NH₂ treated ACH-2 cells, 65% with GPG-NH₂ treated HUT₇₈ cellsand 56% with ALG-NH₂ treated HUT₇₈ cells). ACH-2 cells that were treatedwith 1 mM GPG-NH₂ but not stimulated with PMA to produce virus did notshow such nodules. Combined treatment of PMA stimulated ACH-2 cells withGPG-NH₂ (1 mM) and the protease inhibitor ritonavir (2 μM) gave no suchnodules.

In the latter experiments only budding virus particles of normalappearance and immature virus particles were seen. By tilting, it wasshown that the dense nodules were protruding from the outer cellmembrane. The size which was approximately 50 nm, was that of the dense,distributed material of the irregular viral core which was an internalreference. Occasionally, such a dense nodular structure was alsoobserved attached to the outer part of viral envelope: 2% (with ACH-2cells), 4% (with HUT₇₈ cells) upon GPG-NH₂ treatment and 8% upon ALG-NH₂treatment of HUT₇₈ cells.

In immune EM analysis it was shown that the small particles, assembledon the outer membrane, bound gold-labelled anti-p24 antibody.Furthermore, evaluation of TEM results was accomplished by a 3-Dcomputer modeled reconstruction from tilt TEM of HIV-1 from cultureswith and without GPG-NH₂. These results provide evidence that tripeptideamides inhibit or interfere with viral budding of HIV. The section belowdescribes the use of the small molecules described herein to inhibitreplication of viruses other that HIV.

Small Molecules That Inhibit and/or Prevent Viral Replication andInfection

Small molecules that inhibit viral replication include glycine amide andthe tripeptide amides AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂,LNF-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. Peptidomimeticsthat resemble AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂,GLG-NH₂, and α-t-butylglycine-PG-NH₂ are also embodiments of the presentinvention. The small molecules described herein can be used to inhibitcapsid assembly and replication of viruses that are members of thearenavirus, rotavirus, orbivirus, retrovirus, papillomavirus,adenovirus, herpesvirus, paramyxovirus, myxovirus, and hepadnavirusfamilies. These molecules can be rapidly screened against these viruses,using the teachings described herein or those that would be apparent toone of skill in the art.

To test the ability of glycine amide and the tripeptide amides AIG-NH₂,GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂ to suppress the growth of mammalian DNA virusesanti-viral screening against Herpes Simplex Type 1 (HSV-1) and HerpesSimplex Type 2 (HSV-2) can be performed in tissue culture using humanforeskin fibroblast cells, for example. (See e.g., U.S. Pat. No.6,248,782 to Elford , et al., expressly incorporated by reference in itsentirety). In these infectivity assays, a semi-automated CPE-inhibitionassay can be used employing HSV-1 E-377 strain and HSV-2 MS strain.Additionally, the ability of tripeptide amides to inhibit cytomegalovirus (CMV) can be determined using a semi-automated CPE inhibitionassay and the AD169 strain and for varicellovirus (VZV), a plaquereduction assay using ELLEN strain. Glycine amide and the tripeptideamides can also be screened against Epstein Barr Virus (EBV) in Rajicells (a Burkitt's lymphoma cell line containing 60 EBV genomes/cell)using an immunofluorescence assay with monoclonal antibodies directedagainst EBV components.

Toxicity can be determined by visual inspection of treated cells,generally stationary cells and a cell proliferation assay can be carriedout by determining the presence of rapidly growing cells and either anEC₅₀ (concentration required to inhibit viral cytopathogenicity by 50% )or an IC₅₀ (concentration μg/ml) required to inhibit cell proliferation50% ) can be calculated. Also a Selective Index (S.I.) IC₅₀/EC₅₀ can bedetermined. As above, it is expected that a 100 μM concentration oftripeptide amide or less in these assays would be sufficient tosignificantly inhibit the replication and/or infectivity of HSV, CMV,VZV, and EBV.

It is preferred that the glycine and tripeptides possess a modulationgroup (e.g., an amide group) at their carboxy termini (CO—NH₂) ratherthan a carboxyl group (COOH). Other modulation groups at the carboxyterminus can also be used but desirably, the attached modulation groupshave the same charge and sterically behave the same as an amide group.(See U.S. Pat. No. 5,627,035 to Vahlne et al., for an assay to comparepeptides having differing substituents at the carboxyl terminus, hereinincorporated by reference in its entirety). In some embodiments, theaddition of an acetyl or methyl group at either end of the small peptideis desirable so as to improve uptake of the small peptide or preventexo-protease digestion or both. In the following section, severalapproaches are provided to make biotechnological tools andpharmaceutical compositions comprising the small molecules describedherein.

Biotechnological Tools and Pharmaceutical Compositions ComprisingGlycine Amide and/or Tripeptide Amides

Desirable biotechnological tools or components to prophylactic ortherapeutic agents provide the glycine amide or the tripeptide amides insuch a form or in such a way that a sufficient affinity for inhibitionof a virus, such as HIV-1, is obtained. While a natural monomericpeptide agent (e.g., appearing as discrete units of the peptide agenteach carrying only one binding epitope) is sufficient to bind acapsomere protein, such as p24, and/or interfere with capsid assemblyand/or inhibit proper viral budding and/or prevent viral infection, suchas HIV-1 infection, synthetic ligands or multimeric ligands (e.g.,appearing as multiple units of the peptide agent with several bindingepitopes) may have far greater ability to bind a capsomere protein, suchas p24, and/or interfere with capsid assembly and/or inhibit properviral budding, and/or prevent viral infection, such as HIV-1 infection.It should be noted that the term “multimeric” refers to the presence ofmore than one unit of a ligand, for example several individual moleculesof glycine amide and/or a tripeptide amide, as distinguished from theterm “multimerized” that refers to the presence of more than onetripeptide amide joined as a single discrete unit in tandem.

A multimeric agent (synthetic or natural) that binds a capsomereprotein, such as p24, and/or interferes with capsid assembly and/orinhibits proper viral budding and/or inhibits viral infection, such asHIV-1 infection, may be obtained by coupling glycine amide and/or atripeptide amide to a macromolecular support. The term “support” as usedherein includes a carrier, a resin or any macromolecular structure usedto attach, immobilize, or stabilize a peptide agent. Solid supportsinclude, but are not limited to, the walls of wells of a reaction tray,test tubes, polystyrene beads, magnetic beads, nitrocellulose strips,membranes, microparticles such as latex particles, sheep (or otheranimal) red blood cells, artificial cells and others. The term “support”also includes carriers as that term is understood for the preparation ofpharmaceuticals.

The macromolecular support can have a hydrophobic surface that interactswith a portion of the peptide agent by hydrophobic non-covalentinteraction. The hydrophobic surface of the support can also be apolymer such as plastic or any other polymer in which hydrophobic groupshave been linked such as polystyrene, polyethylene or polyvinyl.Alternatively, the peptide agent can be covalently bound to carriersincluding proteins and oligo/polysaccarides (e.g. cellulose, starch,glycogen, chitosane or aminated sepharose). In these later embodiments,a reactive group on the peptide agent, such as a hydroxy or an aminogroup, can be used to join to a reactive group on the carrier so as tocreate the covalent bond. The support can also have a charged surfacethat interacts with the peptide agent. Additionally, the support canhave other reactive groups that can be chemically activated so as toattach a peptide agent. For example, cyanogen bromide activatedmatrices, epoxy activated matrices, thio and thiopropyl gels,nitrophenyl chloroformate and N-hydroxy succinimide chlorformatelinkages, and oxirane acrylic supports are common in the art.

The support can also comprise an inorganic carrier such as silicon oxidematerial (e.g. silica gel, zeolite, diatomaceous earth or aminatedglass) to which the peptide agent is covalently linked through ahydroxy, carboxy or amino group and a reactive group on the carrier.Furthermore, in some embodiments, a liposome or lipid bilayer (naturalor synthetic) is contemplated as a support and peptide agents areattached to the membrane surface or are incorporated into the membraneby techniques in liposome engineering. By one approach, liposomemultimeric supports comprise a peptide agent that is exposed on thesurface of the bilayer and a second domain that anchors the peptideagent to the lipid bilayer. The anchor can be constructed of hydrophobicamino acid residues, resembling known transmembrane domains, or cancomprise ceramides that are attached to the first domain by conventionaltechniques.

Supports or carriers for use in the body, (i.e. for prophylactic ortherapeutic applications) are desirably physiological, non-toxic andpreferably, non-immunoresponsive. Contemplated carriers for use in thebody include poly-L-lysine, poly-D, L-alanine, liposomes, andChromosorb® (Johns-Manville Products, Denver Colo.). Ligand conjugatedChromosorb® (Synsorb-Pk) has been tested in humans for the prevention ofhemolytic-uremic syndrome and was reported as not presenting adversereactions. (Armstrong et al. J. Infectious Diseases, 171:1042–1045(1995)). For some embodiments, the administration of a “naked” carrier(i.e., lacking an attached peptide agent) that has the capacity toattach a peptide agent in the body of a subject is contemplated. By thisapproach, a “prodrug-type” therapy is envisioned in which the nakedcarrier is administered separately from the peptide agent and, once bothare in the body of the subject, the carrier and the peptide agent areassembled into a multimeric complex.

Additionally, prodrugs, which are compounds that break down in the body(e.g., a human) to yield an active ingredient of the invention (e.g.,glycine amide or a tripeptide selected from the group consisting ofAIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂) areembodiments. It is contemplated that several molecules can be designedsuch that upon introduction to a human, they undergo proteolysis ordegradation to achieve glycine amide or a tripeptide selected from thegroup consisting of AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂,APG-NH₂, GLG-NH₂. Because these prodrug molecules break down to theactive ingredients glycine amide or a tripeptide selected from the groupconsisting of AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂,GLG-NH₂ they are equivalent to these molecules.

The insertion of linkers, such as λ linkers, of an appropriate lengthbetween the peptide agent and the support are also contemplated so as toencourage greater flexibility of the peptide agent and thereby overcomeany steric hindrance that may be presented by the support. Thedetermination of an appropriate length of linker that allows for optimalbinding to a capsomere protein, such as p24, and/or interference withcapsid assembly and/or inhibition of viral infection, such as HIVinfection, can be determined by screening the peptide agents withvarying linkers in the assays detailed in the present disclosure.

Another aspect of the invention includes a composite support comprisingmore than one type of peptide agent. A “composite support” may be acarrier, a resin, or any macromolecular structure used to attach orimmobilize two or more different peptide agents that bind to a capsomereprotein, such as p24, and/or interfere with capsid assembly and/orinhibit proper viral budding and/or inhibit viral infection, such as HIVinfection. In some embodiments, a liposome or lipid bilayer (natural orsynthetic) is contemplated for use in constructing a composite supportand peptide agents are attached to the membrane surface or areincorporated into the membrane using techniques in liposome engineering.

The insertion of linkers, such as λ linkers, of an appropriate lengthbetween the peptide agent and the support is also contemplated so as toencourage greater flexibility in the molecule and thereby overcome anysteric hindrance that may occur. The determination of an appropriatelength of linker that allows for optimal binding to a capsomere protein,such as p24, and/or interference with capsid assembly and/or inhibitionof proper viral budding and/or inhibition of viral infection, such asHIV infection, can be determined by screening the ligands with varyinglinkers in the assays detailed in the present disclosure.

In other embodiments, the multimeric and composite supports discussedabove can have attached multimerized ligands so as to create a“multimerized-multimeric support” and a “multimerized-compositesupport”, respectively. A multimerized ligand can be obtained, forexample, by coupling two or more peptide agents in tandem usingconventional techniques in molecular biology. The multimerized form ofthe ligand can be advantageous for many applications because of theability to obtain an agent with a better ability to bind to a capsomereprotein, such as p24, and/or interfere with capsid assembly and/orinhibit viral infection, such as HIV or SIV infection. Further, theincorporation of linkers or spacers, such as flexible λ linkers, betweenthe individual domains that make-up the multimerized agent is anotherembodiment. The insertion of λ linkers of an appropriate length betweenprotein binding domains, for example, can encourage greater flexibilityin the molecule and can overcome steric hindrance. Similarly, theinsertion of linkers between the multimerized ligand and the support canencourage greater flexibility and limit steric hindrance presented bythe support. The determination of an appropriate length of linker thatallows for optimal binding to p24 and/or interference with capsidassembly and/or inhibition of proper viral budding and/or inhibition ofHIV infection, can be determined by screening the ligands with varyinglinkers in the assays detailed in this disclosure.

In preferred embodiments, the various types of supports discussed aboveare created using glycine amide or the tripeptide amides AIG-NH₂,GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂. The multimeric supports, composite supports,multimerized-multimeric supports, or multimerized-composite supports,collectively referred to as “support-bound agents”, are also preferablyconstructed using glycine amide or the tripeptide amides AIG-NH₂,GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂.

The monomeric and multimeric peptide agents described herein aresuitable for use as a biotechnological tool to study the interaction ofglycine amide or tripeptide amides with capsid proteins and also asmedicaments for the treatment of subjects already infected with a virus,such as HIV, or as a preventive measure to avoid viral infections, suchas HIV infection. Although anyone could be treated with glycine amide orthe tripeptide amides as a prophylactic, the most suitable subjects arepeople at risk for viral infection. Such subjects include, but are notlimited to, the elderly, the chronically ill, homosexuals, prostitutes,intravenous drug users, hemophiliacs, children, and those in the medicalprofession who have contact with patients or biological samples. Thefollowing section discusses methods of making and using the medicamentsdescribed herein.

Methods of Making and Using Medicaments Comprising Glycine Amide and/orTripeptide Amides

Methods of making and using medicaments comprising glycine amide and/orthe tripeptide amides disclosed herein are also embodiments of thepresent invention. The peptide agents described herein can be processedin accordance with conventional methods of galenic pharmacy to producemedicinal agents for administration to patients, e.g., mammals includinghumans. The peptide agents can be incorporated into a pharmaceuticalproduct with and without modification. Further, the manufacture ofpharmaceuticals or therapeutic agents that deliver the peptide agent byseveral routes is included within the scope of the present invention.

The compounds described herein can be employed in admixture withconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral (e.g.,oral) or topical application that do not deleteriously react with thepeptide agents. Suitable pharmaceutically acceptable carriers include,but are not limited to, water, salt solutions, alcohols, gum arabic,vegetable oils, benzyl alcohols, polyethylene glycols, gelatine,carbohydrates such as lactose, amylose or starch, magnesium stearate,talc, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like that do notdeleteriously react with the active compounds.

In some embodiments, medicaments comprising glycine amide and/ortripeptide amides are formulated with or administered in conjunctionwith other agents that inhibit viral infections, such as HIV infection,so as to achieve a better viral response. At present four differentclasses of drugs are in clinical use in the antiviral treatment of HIV-1infection in humans. These are (i) nucleoside analogue reversetranscriptase inhibitors (NRTIs), such as zidovudine, lamivudine,stavudine, didanosine, abacavir, and zalcitabine; (ii) nucleotideanalogue reverse transcriptase inhibitors, such as adetovir and pivaxir;(iii) non-nucleoside reverse transcriptase inhibitors (NNRTIs), such asefavirenz, nevirapine, and delavirdine; and (iv) protease inhibitors,such as indinavir, nelfinavir, ritonavir, saquinavir and amprenavir. Bysimultaneously using two, three, or four different classes of drugs inconjunction with administration of the peptide agents, HIV is lesslikely to develop resistance, since it is less probable that multiplemutations that overcome the different classes of drugs and the peptideagents will appear in the same virus particle.

It is thus preferred that medicaments comprising peptide agents (e.g.,glycine amide or a tripeptide amide selected from the group consistingof AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂) be formulated with or given in combination withnucleoside analogue reverse transcriptase inhibitors, nucleotideanalogue reverse transcriptase inhibitors, non-nucleoside reversetranscriptase inhibitors, and protease inhibitors at doses and bymethods known to those of skill in the art. Medicaments comprising thepeptide agents and nucleoside analogue reverse transcriptase inhibitors,nucleotide analogue reverse transcriptase inhibitors, non-nucleosidereverse transcriptase inhibitors, and protease inhibitors can beformulated to contain other ingredients to aid in delivery, retention,or stability of the glycine amide and/or the tripeptide amide.

The effective dose and method of administration of a particular peptideagents formulation can vary based on the individual patient and thestage of the disease, as well as other factors known to those of skillin the art. Therapeutic efficacy and toxicity of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED₅₀ and LD₅₀ (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors that may be taken into account include theseverity of the disease state, age, weight and gender of the patient;diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to therapy. Short actingpharmaceutical compositions are administered daily whereas long actingpharmaceutical compositions are administered every 2, 3 to 4 days, everyweek, or once every two weeks. Depending on half-life and clearance rateof the particular formulation, the pharmaceutical compositions of theinvention are administered once, twice, three, four, five, six, seven,eight, nine, ten or more times per day.

Normal dosage amounts may vary from approximately 1 to 100,000micrograms, up to a total dose of about 20 grams, depending upon theroute of administration. Desirable dosages include 250 μg, 500 μg, 1 mg,50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg,500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg,1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g,3 g, 4 g, 5, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16g, 17 g, 18 g, 19 g, and 20 g. Additionally, the concentrations of thepeptide agents can be quite high in embodiments that administer theagents in a topical form. Molar concentrations of peptide agents can beused with some embodiments. Desirable concentrations for topicaladministration and/or for coating medical equipment range from 100 μM to800 mM. Preferable concentrations for these embodiments range from 500μM to 500 mM. For example, preferred concentrations for use in topicalapplications and/or for coating medical equipment include 500 μM, 550μM, 600 μM, 650 μM, 700 μM, 750 μM, 800 μM, 850 μM, 900μM, 1 mM, 5 mM,10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 60 mM, 70mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170mM, 180 mM, 190 mM, 200 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425mM, 450 mM, 475 mM, and 500 mM. Guidance as to particular dosages andmethods of delivery is provided in the literature and below. (See e.g.,U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212, herein expresslyincorporated by reference in their entireties).

More specifically, the dosage of the peptide agents described herein isone that provides sufficient peptide agent to attain a desirable effectincluding binding of a capsomere protein, such as p24, and/orinterference with capsid assembly and/or inhibition of proper viralbudding and/or inhibition of viral infection, such as HIV infection.Accordingly, the dose of peptide agent preferably produces a tissue orblood concentration or both from approximately 0.1 nM to 500 mM.Desirable doses produce a tissue or blood concentration or both of about0.1 nM to 800 μM. Preferable doses produce a tissue or bloodconcentration of greater than about 10 nM to about 300 μM. Preferabledoses are, for example, the amount of molecule required to achieve atissue or blood concentration or both of 10 nM, 15 nM, 20 nM, 25 nM, 30nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80nM, 85 nM, 90 nM, 95 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM,700 nM, 800 nM, 900 nM, 1 μM, 10 μM, 50 μM, 100 μM, 200 μM, and 300 μM.Although doses that produce a tissue concentration of greater than 800μM are not preferred, they can be used with some embodiments. A constantinfusion of the glycine amide and/or tripeptide amide can also beprovided so as to maintain a stable concentration in the tissues asmeasured by blood levels.

Routes of administration of the peptide agents include, but are notlimited to, topical, transdermal, parenteral, gastrointestinal,transbronchial, and transalveolar. Topical administration isaccomplished via a topically applied cream, gel, rinse, etc. containinga tripeptide amide and/or glycine amide. Transdermal administration isaccomplished by application of a cream, rinse, gel, etc. capable ofallowing the peptide agent to penetrate the skin and enter the bloodstream. Parenteral routes of administration include, but are not limitedto, electrical or direct injection such as direct injection into acentral venous line, intravenous, intramuscular, intraperitoneal orsubcutaneous injection. Gastrointestinal routes of administrationinclude, but are not limited to, ingestion and rectal. Transbronchialand transalveolar routes of administration include, but are not limitedto, inhalation, either via the mouth or intranasally.

Compositions of peptide agents containing compounds suitable for topicalapplication include, but are not limited to, physiologically acceptableimplants, ointments, creams, rinses, and gels. Any liquid, gel, or solidpharmaceutically acceptable base in which the peptides are at leastminimally soluble is suitable for topical use in the present invention.Compositions for topical application are particularly useful duringsexual intercourse to prevent transmission of HIV. Suitable compositionsfor such use include, but are not limited to, vaginal or analsuppositories, creams, and douches.

Compositions of the peptide agents suitable for transdermaladministration include, but are not limited to, pharmaceuticallyacceptable suspensions, oils, creams, and ointments applied directly tothe skin or incorporated into a protective carrier such as a transdermaldevice (“transdermal patch”). Examples of suitable creams, ointments,etc. can be found, for instance, in the Physician's Desk Reference andare well known in the art. Examples of suitable transdermal devices aredescribed, for instance, in U.S. Pat. No. 4,818,540, issued Apr. 4, 1989to Chinen, et al., hereby incorporated by reference in its entirety.

Compositions of the peptide agents suitable for parenteraladministration include, but are not limited to, pharmaceuticallyacceptable sterile isotonic solutions. Such solutions include, but arenot limited to, saline and phosphate buffered saline for injection intoa central venous line, intravenous, intramuscular, intraperitoneal, orsubcutaneous injection of the peptide agents.

Compositions of the peptide agents suitable for transbronchial andtransalveolar administration include, but are not limited to, varioustypes of aerosols for inhalation. For instance, pentamidine isadministered intranasally via aerosol to AIDS patients to preventpneumonia caused by pneumocystis carinii. Devices suitable fortransbronchial and transalveolar administration of the peptides,including but not limited to atomizers and vaporizers, are also includedwithin the scope of the present invention. Many forms of currentlyavailable atomizers and vaporizers can be readily adapted to deliverpeptide agents.

Compositions of the peptide agents suitable for gastrointestinaladministration include, but not limited to, pharmaceutically acceptablepowders, pills, sachets, or liquids for ingestion and suppositories forrectal administration. Due to the most common routes of HIV infectionand the ease of use, gastrointestinal administration, particularly oral,is the preferred embodiment of the present invention. Five-hundredmilligram capsules having a tripeptide amide have been prepared and werefound to be stable for a minimum of 12 months when stored at 4° C. Sincesmall peptides apparently evade degradation by the patient's digestivesystem, they are ideal for oral administration.

The peptide agents are also suitable for use in situations whereprevention of HIV infection is important. For instances, medicalpersonnel are constantly exposed to patients who may be HIV positive andwhose secretions and body fluids contain the HIV virus. Further, thepeptide agents can be formulated into antiviral compositions for useduring sexual intercourse so as to prevent transmission of HIV. Suchcompositions are known in the art and also described in theinternational application published under the PCT publication numberWO90/04390 on May 3, 1990 to Modak et al., which is incorporated hereinby reference in its entirety.

Embodiments of the invention also include a coating for medicalequipment such as gloves, sheets, and work surfaces that protectsagainst viral transmission. Alternatively, the peptide agents can beimpregnated into a polymeric medical device. Particularly preferred arecoatings for medical gloves and condoms. Coatings suitable for use inmedical devices can be provided by a powder containing the peptides orby polymeric coating into which the peptide agents are suspended.Suitable polymeric materials for coatings or devices are those that arephysiologically acceptable and through which a therapeutically effectiveamount of the peptide agent can diffuse. Suitable polymers include, butare not limited to, polyurethane, polymethacrylate, polyamide,polyester, polyethylene, polypropylene, polystyrene,polytetrafluoroethylene, polyvinyl-chloride, cellulose acetate, siliconeelastomers, collagen, silk, etc. Such coatings are described, forinstance, in U.S. Pat. No. 4,612,337, issued Sep. 16, 1986 to Fox etal., which is incorporated herein by reference in its entirety.

Accordingly, methods of making a medicament that inhibits viralreplication, specifically, HIV, involve providing glycine amide and/or atripeptide amide selected from the group consisting of AIG-NH₂, GFG-NH₂,GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂, and formulating said medicament for delivery toa subject, including a human, as described above.

Methods of identification of peptide agents that inhibit viralreplication, specifically HIV replication, are also provided. By onemethod, for example, a peptide agent for incorporation into ananti-viral pharmaceutical is identified by contacting a plurality ofcells infected with a virus with an effective amount of a peptide agentanalyzing the virus for incomplete capsid formation and/or impairedviral budding, and selecting the peptide agent that induces incompletecapsid formation. This method can involve microscopic analysis and thevirus can be selected from the group consisting of HIV-1, HIV-2, andSIV. Further, the peptide agent identified can be selected from thegroup consisting of glycine amide, a tripeptide amide, and apeptidomimetic resembling a tripeptide amide. For example, the peptideagent above can be selected from the group consisting of glycine amide,AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, andα-t-butylglycine-PG-NH₂.

In another embodiment, a method of identifying a peptide agent thatbinds to a viral protein is provided. Some aspects of this methodinvolve providing a viral protein, contacting the viral protein with aneffective amount of a peptide agent, and detecting the formation of acomplex comprising the viral protein and the peptide agent. Preferably,the viral protein is from a virus selected from the group consisting ofHIV-1, HIV-2, and SIV. The detection step can be accomplished byperforming a binding assay (e.g., a p24 binding assay involvingdialysis, capillary electrophoresis, computer modeling, orcrystallography).

A method of identifying a peptide agent that binds to a viral proteinusing dialysis can be performed, as follows. Approximately 50 μl of 10μM solutions of recombinant protein p24, recombinant gp120, or BSA areplaced in a 10 kD cut-off dialysis cassette (Slide-A-Lyzer from Pierce)and are dialyzed against 500 ml of buffer containing 150 mM NaCl, 50 mMTris-HCl, pH 7.4, and 27.5 μM ¹⁴C labeled tripeptide amide at 4° C. for2 days. Radioactivity can then be quantified in a Rackbeta 1218 (LKBWallace) after mixing 10 μl or 5 μl of the proteins in ReadySafe(Beckman).

Another method of identifying a peptide agent that binds to a viralprotein using dialysis can be performed as follows. A piece of fusedsilica tubing (inner diameter 50 μm) is cut to a length of 23 cm (lengthto the detector 18.5 cm) and coated prior to use with 5% (w/v) linearpolyacrylamide (Hjertén, S. J. Chromatogr. 347, 191–198 (1985),expressly incorporated by reference in its entirety) in order tosuppress the electroendosmotic flow and to avoid unwanted adsorption ofproteins onto the capillary wall. A 0.01 M sodium phosphate solution isused as buffer in the pH range 6.8–8.2. The tripeptide amides aredissolved in the buffer at a relatively high concentration (0.5 mg/ml)because of their low UV-absorbance. A stock solution of p24 is dilutedten-fold with the running buffer to a final concentration of 50 μg/ml.The capillary is filled with the buffer. The protein is injected bypressure (50 psi per second) and then the tripeptide sample (1 psi persecond). Since the electrophoretic migration velocity of the peptide ishigher than that of the protein, the peptide molecules will move throughthe protein zone (Hjertén, S. Analysis and purification of cells withthe free zone electrophoresis equipment. In Cell Separation Methods,Loemendal, H., editor. Elsevier/North-Holland Biomedical Press (1977),expressly incorporated by reference in its entirety). Spectra can berecorded over the whole UV range (195–360 nm with 5 nm frequency) for ontube identification of the peaks. An interaction between the viralprotein and the peptide will be revealed as an increase in migrationtime of the peptide compared to that in the absence of the protein.

In some embodiments, the peptide agent is selected from the groupconsisting of glycine amide, AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂,GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. Additionally, amethod of making a pharmaceutical is provided in which the peptide agentidentified by the methods above are incorporated in a pharmaceutical.

Another approach to making a pharmaceutical involves administering to acell an effective amount of glycine amide and/or a tripeptide amide,described above, detecting an inhibition of viral replication in thecell, and incorporating the tripeptide amide that causes inhibition ofviral replication into the pharmaceutical. This method can involve theuse of glycine amide or a tripeptide amide selected from the groupconsisting of AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂,GLG-NH₂, and α-t-butylglycine-PG-NH₂. Further, the method above can besupplemented with administration of an antiviral compound selected fromthe group consisting of nucleoside analogue reverse transcriptaseinhibitors, nucleotide analogue reverse transcriptase inhibitors,non-nucleoside reverse transcriptase inhibitors, and protease inhibitorsinto the pharmaceutical. Additionally, the method above can besupplemented by incorporating a carrier into the pharmaceutical.

Although the peptide agents described herein can be used as researchtools to analyze the interaction of glycine amide and/or a tripeptideamide with a protein, desirably they are used to inhibit viralreplication and/or infection, preferably, HIV replication and infectionin a subject. By one method, for example, a subject at risk of becominginfected by HIV or who is already infected with HIV is identified andsaid subject is provided glycine amide and/or a tripeptide amideselected from the group consisting of AIG-NH₂, GFG-NH₂, GWG-NH₂,FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. By anadditional method, a subject is provided glycine amide and/or atripeptide amide selected from the group consisting of AIG-NH₂, GFG-NH₂,GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂and the effect on viral replication or infection, preferably HIVreplication or infection, is determined (e.g., by analyzing the amountof p24 or reverse transcriptase activity in a sample).

The methods above can be supplemented with administration of anantiviral treatment selected from the group consisting of nucleosideanalogue reverse transcriptase inhibitors, nucleotide analogue reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and protease inhibitors. Further, the tripeptide amide usedin these methods can be joined to a support or can be administered in apharmaceutical comprising a pharmaceutically acceptable carrier.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures and tables,as well as patents, applications, and publications referred to above arehereby expressly incorporated by reference in their entireties.

1. An antiviral composition for human administration comprising monopeptidic glycine amide and a compound that inhibits replication of HIV in the presence of monopeptidic glycine amide.
 2. The antiviral composition of claim 1, wherein said antiviral composition is formulated for oral administration.
 3. The antiviral composition of claim 1, wherein the amount of glycine amide is 250 μg or more.
 4. The antiviral composition of claim 1, wherein said compound that inhibits replication of HIV in the presence of monopeptidic glycine amide is a peptide.
 5. The antiviral composition of claim 4, wherein said peptide is selected from the group consisting of AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and a-t-butylglycine-PG-NH₂.
 6. The antiviral composition of claim 4, wherein said antiviral composition is formulated for oral administration.
 7. The antiviral composition of claim 4, wherein said antiviral composition comprises 250 μg glycine amide.
 8. The antiviral composition of claim 4, wherein said antiviral composition comprises 1 mg glycine amide.
 9. The antiviral composition of claim 4, wherein said antiviral composition comprises 50 mg glycine amide.
 10. The antiviral composition of claim 4, wherein said antiviral composition comprises 100 mg glycine amide.
 11. The antiviral composition of claim 4, wherein said antiviral composition comprises 300 mg glycine amide.
 12. The antiviral composition of claim 1, wherein said compound that inhibits replication of HIV in the presence of monopeptidic glycine amide is selected from the group consisting of nucleoside analogue reverse transcriptase inhibitors, nucleotide analogue reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors.
 13. The antiviral composition of claim 12, wherein said compound that inhibits replication of HIV in the presence of monopeptidic glycine amide is selected from the group consisting of azidothymidine (AZT), zidovudine, lamivudine, stavudine, didanosine, abacavir, zalcitabine, adetovir, pivaxir, efavirenz, nevirapine, delavirdine, indinavir, nelfinavir, ritonavir, saquinavir, and amprenavir.
 14. The antiviral composition of claim 12, wherein said antiviral composition is formulated for oral administration.
 15. The antiviral composition of claim 12, wherein the amount of glycine amide is 250 μg or more.
 16. A pill comprising monopeptidic glycine amide formulated for human administration in unit dosage form.
 17. An oral suspension comprising 1 mg monopeptidic glycine amide and a flavoring, formulated for human administration.
 18. A suppository comprising monopeptidic glycine amide formulated for human administration.
 19. A transdermal composition comprising monopeptidic glycinamide formulated for human administration. 